Epithelial membranes act as the interface between tissue compartments. Transport of macromolecules across this barrier is a critical process in most tissues, particularly the gastrointestinal tract where antigens are sampled from the intestinal lumen and secretory antibodies transported from the lamina propria into mucosal secretions. Epithelial transport is mediated by vesicle carriers, whose formation and targeting are highly regulated and specific. Key components of the vesicular transport machinery are small GTPases of the RAS superfamily. These proteins appear to act as molecular switches, controlling the assembly, targeting, and fusion of organelle-specific carrier vesicles. Among these are the ADP- ribosylation factors (ARFs). Unlike other members of the RAS family, ARFs have no intrinsic GTPase activity, and therefore require the cooperation of a GTPase activating protein (GAP). Six mammalian ARFs have been reported which, although highly homologous, appear to have distinct functions within cells. While ARF1 functions in the assembly of carrier vesicles in the endoplasmic reticulum and Golgi, ARF6, the subject of this proposal, appears to function in the endocytic pathway. In contrast to non-polarized cells, epithelial cells have distinct endocytic pathways at both apical and basolateral poles, and transcytotic pathways that communicate between them. The overall aim of this proposal is to define the role of ARF6 in polarized epithelial cells, and the intracellular compartments to which it localizes. Additionally, we propose to determine the functional consequences of a panel of mutants on the cell's endocytic and transcytotic processes, and attempt detection and characterization of an ARF6-specific GAP.